Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• IGF-I analogs described in this invention are produced as single chain IGF-I-like molecules with equal potency to IGF-I at the type I IGF receptor and reduced binding to 28 K IGF Binding Proteins rendering such analogs of significant potential in vivo utility.
• hIGF-I Human insulin-like growth factor I
• somatomedin C Human insulin-like growth factor I
• Human IGF-I shows a remarkable amino acid sequence homology to insulin. This homology is the basis of a computer generated three-dimensional structural model for hIGF-I. (Blundell et al. Proc. Natl. Acad. Sci. U.S.A. 75 : 180-184 (1978) and Blundell et al. Fed. Proc. Am. Soc. Exp. Biol. 42 : 2592-2597 (1983)). This model predicts that a portion of the insulin receptor binding region is conserved within the IGF-I molecule explaining the ability of hIGF-I to bind to insulin receptors. The model also suggests regions of hIGF-I molecule which may be responsible for binding to serum carrier proteins.
• IGF-I insulin-binding protein
• serum carrier proteins which do not readily cross the capillary barrier.
• IGF I insulin-binding Protein
• IGF I-Binding Protein 28 K IGF Binding Protein which is present in serum and amniotic fluid and which is secreted by most cells.
• binding proteins clearly play a role in modulating the activity of IGF.
• the presence of serum binding proteins is a barrier to the bioactivity and bioavailability of exogenously administered IGF-I.
• IGF-I insulin-binds to these proteins.
• a synthetic gene for human IGF-I was modified to encode two IGF-I analogs in which residues 42 to 58 of hIGF-I are replaced by the first 17 amino acids of the A chain of human insulin and in which residues 49-51 of hIGF I are replaced by residues 8-10 of the A chain of human insulin.
• the synthetic genes are then placed in a yeast recombinant DNA expression system and the peptide analogs which are produced by the modified yeast cells are extracted therefrom and purified.
• IGF150 and IGF125 contain the first 17 amino acids of human insulin A chain in place of residues 42 to 58 of hIGF-I, and residues 8-10 of the A chain of insulin in place of residues 49-51 of hIGF-I, respectively. These analogs have near equal affinity for the type I IGF receptor as compared to normal human IGF-I (Table 1). However, analogs IGF150 and IGF 125, have reduced binding to both human and rat 28K IGF binding proteins (Table 2). Thus, these new proteins retain nearly full activity at the type I IGF receptor but have reduced binding to 28 K IGF binding proteins. It is expected that these analogs will be more potent in vivo than normal IGF-I. IGF150 is 100 times more potent than normal IGF-I in stimulating DNA synthesis in 3T3 cells ( Figure 4).
• the synthetic genes of this invention encode for peptides which are analogs of human insulin-like growth factor (hIGF-I) and have the following structure where the letter designation for the constituent amino acids have the definitions given below: IGF(1-40)-A1-GIV-A2-A3-C-C-A4-A5-A6-C-A7-L-A8-A9-LE-R wherein: A1 is T or I; A2 is D or E; A3 is E or Q; A4 is T; A5 is S; A6 is I; A7 is D or S; A8 is R or Y; A9 is R or Q; and R is the remainder of the hIGF-I peptide consisting of 12 amino acids as follows: MYCAPLKPAKSA with the exception that the following gene: IGF(1-40)-TGVIDECCFRSCDLRRLE-R which is the wild type hIGF-I and is excluded from the foregoing definition.
• IGF(1-40)IGIVEQCCTSICSLYQLE-R Compound A or IGF150
• IGF(1-40)TGIVDECCTSICDLRRLE-R Compound B or IGF125
• the peptide analogs can be produced by procedures similar to methods existing for the preparation of natural hIGF-I peptide, and modifications thereof which would be well-known to those skilled in the art. Specifically, these analogs may be synthesized chemically using procedures developed for human IGF-I. See for example Li et al. Proc. Natl. Acad. Sci. U.S.A. 80 : 2216-2220 (1983). In accordance with the present invention the IGF-I analogs may also be produced following the transformation of susceptible bacterial, yeast or tissue culture cell hosts with recombinant plasmids that include DNA sequences capable of directing the expression of IGF-I analogs.
• the DNA sequence may be prepared synthetically, chromosomally, by recombinant DNA techniques or combination thereof.
• DNA sequences capable of directing the expression of IGF-I analogs could also be introduced into the germ line of animals or extra-chromasomally to produce transgenic animals endogenously producing the IGF-I analogs.
• the instant synthetic gene produces analogs of hIGF-I which have substantial activity but, because they do not bind to 28 K IGF binding protein have levels of activity which, when taken on a molar or weight basis are considerably more active than wild-type hIGF-I.
• the compounds are thus highly active as agents to increase the yield and efficency of milk production of animals, particularly ruminant animals such as cows.
• the compounds are also useful as growth promotant agents in food producing animals by increasing the rate of gain, feed efficency and carcass quality.
• the compounds are further useful as agents to promote wound healing and to stimulate erythropoiesis (the manufacture of red blood cells).
• the compounds When used to increase milk production or as an animal growth promotant the compounds are administered parenterally such as by subcutaneous, intramuscular or intravenous injection or by a sus­ tained release subcutaneous implant.
• the active ingredient In subcutaneous, intramuscular and intravenous injection the active ingredient is dissolved or dispersed in a liquid carrier vehicle.
• the active material is suitably admixed with an accept­ able vehicle, preferably of the vegetable oil variety such as peanut oil, cotton seed oil and the like.
• Other parenteral vehicles such as organic preparation using solketal, glycerol, formal and aqueous parenteral formulations are also used.
• the active compound or compounds are dissolved or suspended in the parenteral formulation for administration; such formulations generally contain from 0.005 to 5% by weight of the active compound.
• the instant compounds are effective by significantly increasing the level of milk production or the rate of weight gain or feed efficiency when administered at levels of from 0.1 to 100 mg per kg of animal body weight, preferably at from 1 to 10 mg/kg.
• the compounds are administered in the form of a subcutaneous implant the compound is suspended or dissolved in a slowly dispersed material known to those skilled in the art, or administered in a device which slowly releases the active material through the use of a constant driving force such as an osmotic pump. In such cases constant administration over periods ranging from 20 to 120 days are possible with the active ingredient being released at from 0.1 to 10 mg/kg/day.
• the hIGF-I analogs act synergistic strictly ally with platelet-derived growth factor (PDGF) or other competence factors such as fibroblast growth factor (FGF) to stimulate DNA synthesis and cell replication in human fibroblasts, such analogs are useful to promote wound healing especially in cases where endogenous hIGF levels are low.
• PDGF platelet-derived growth factor
• FGF fibroblast growth factor
• the instant IGF-I analogs may be administered in combination with PDGF or FGF.
• the compounds could be administered parenterally, either subcutaneously, intramuscularly or intravenously using pharmaceutically acceptable parenteral formulation ingredients such as those listed above.
• the compounds would be administered at a dose of from 0.1 to 100 mg/kg, preferably from 1 to 10 mg/kg.
• the compounds are administered topically when used as an agent to promote wound healing.
• Typical formulations for topical application are liquid, paste, ointment and spray formulations.
• the formulations could also be incorporated into a dressing which would be applied to the wound. The dressing would slowly release the compound directly to the site needing treatment.
• the compounds would be incorporated into the topical formulation at concentrations of from 0.003 to 10% by weight with most formulations requiring from 0.3 to 3%.
• the concentration could be adjusted to provide for daily doses of from 0.06 to 2 mg of the active compound with allowance made to provide for multiple applications during any particular day.
• the instant compounds may also be useful as erythropoietic agents possibly by virtue of their ability to stimulate late erythroid precursor differentiation.
• the compounds are administered parenterally as described above.
• the compounds may be administered either alone or in combination with erythropoietin to promote the production of red blood cells.
• the compounds are administered at doses of from 0.1 to 100 mg/kg, preferably from 1 to 10 mg/kg. Such doses are on a daily basis and if needed, the dose may be divided into multiple daily doses.
• Plasmid phIGF was modified to form plasmid pJY2 (Bayne et al Gene 66 235-244(1988)). Plasmid pJY2 was modified as described in Figure 1.
• the Bam HI IGF150 gene cassette from plasmid pJY150 was ligated into Bam HI digested p ⁇ 2 as indicated in Figure 1.
• the plasmid with the IGF150 cassette in p ⁇ 2 in the proper orientation was designated p ⁇ 2IGF150.
• This plasmid was introduced into the yeast strain BJ1995. Yeast strain carrying the p ⁇ 2IGF150 plasmid secrete the protein IGF150 into the growth media.
• Saccharomyces cerevisiae strain BJ1995 (MAT ⁇ , leu2, trp1, ura3, prb1-1122, pep4-3, ciro) was transformed with the appropriate expression plasmid and transformants were selected on leucine minus plates.
• Cells were grown to saturation in 1 liter of 5x leu(-) media, pH 4.8, containing 0.85% yeast nitrogen base without amino acids and ammonium sulfate supplemented with 4% glucose, 1% ammonium sulfate, 0.6% sodium hydroxide, 0.03% L-isoleucine, 0.03% L-phenylalanine, 0.025% L-tyrosine, 0.02% L-lysine, 0.02% L-tryptophan, 0.02% uracil, 0.02% adenine, 0.01% L-arginine, 0.005% methionine, 0.005% L-histidine, 29 ⁇ M ferric chloride, 25 ⁇ M zinc sulfate, and 1% succinic acid.
• Quantitative amino acid analysis was employed to determine the concentration of purified analogs.
• the amino acid composition is consistent with that expected for the analogs.
• Affinity of the analogs to type I IGF receptor is shown in Table 1.
• Analog A (IGF150), and B (IGF125) inhibit the binding of 123I-hIGF-I to human placental membranes with a IC50 of 3.2 nM, and 7.0 nM respectively, compared to 4.9 nM for wild type recombinant hIGF-I.
• Affinity of analogs A and B for human and murine 28 K IGF binding human proteins are shown in Table 2.
• Recombinant wild type hIGF-I inhibits binding of 123-hIGF-I to the 28 K human and murine IGF binding proteins with a IC50 of 0.23 nM and 11.2 nM, respectively.
• Analog IGF150 inhibits this binding with a IC50 > 1.5 nM and 71 nM, respectively.
• Analog IGF125 inhibits this binding with IC50»1.5 nM and 501 nM, respectively.
• IGF-I stimulates DNA synthesis in mouse 3T3 cells. As shown in Figure 4, IGF150 stimulates DNA synthesis in these cells with about 100-fold higher potency then wild type IGF-I.

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• 1990
  • 1990-01-16 CA CA002007886A patent/CA2007886A1/fr not_active Abandoned
  • 1990-01-16 EP EP19900300450 patent/EP0379338A3/fr not_active Withdrawn
  • 1990-01-17 JP JP2006515A patent/JPH0367589A/ja active Pending

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